Genetics of Cystic Fibrosis

Genetics of Cystic Fibrosis

Learning Outcomes

• Discuss cystic fibrosis as an example of the successes and limitations in the diagnosis and treatment of a rare genetic disease.
• Describe the differences between different mutations and disease mechanisms.
• Understand the importance of genotype/phenotype correlation.
• Discuss approaches to genetic therapies.

Cystic Fibrosis: From Mutation to Phenotype to Therapy

• Defective CF gene leads to:
  • Deficient CFTR protein.
  • Decreased chloride secretion.
  • Altered ionic transport (Chloride, Sodium).
  • Increased water absorption.
  • Abnormal mucus composition.
  • Bronchial obstruction.
  • Bacterial infections.
  • Inflammation.
  • Bronchiectasis and lung insufficiency.

Clinical Diagnosis

• Sweat test to measure chloride concentration:
  • 0 - 29 mmol/L: CF is unlikely.
  • 30 - 59 mmol/L: CF is possible; additional testing needed.
  • >= 60 mmol/L: CF is likely.
• Example Case: Joanne Brown had a sweat test with [Cl-] = 87 mmol/L.

Cystic Fibrosis as a Pleiotropic Disease

• Affects multiple organs with varying phenotypes:
  • LUNG: Abnormal mucus leading to infection and damage.
  • GASTROINTESTINAL TRACT: Meconium ileus (neonatal bowel obstruction), intestinal obstruction.
  • PANCREAS: Pancreas dysfunction, malabsorption.
  • HEPATOBILIARY TRACT: Biliary cirrhosis, gallstones.
  • SWEAT GLAND: Elevated chloride and sodium in sweat.
  • REPRODUCTIVE TRACT: Absence of vas deferens, thick cervical secretions.

Family History and Molecular Testing

• Mode of inheritance: De novo or autosomal recessive?
• Reasons to do a molecular test:
  • Clinical diagnosis and lab findings secure in most cases.
  • Check for subtypes and clinical heterogeneity.
  • Determine carrier status and offer prenatal testing.
  • Important for prognosis and therapy.

Genetics of Cystic Fibrosis

• Lethal autosomal recessive disorder (with recent improvements in life expectancy).
• UK prevalence: 1 in 2,500 newborns.
• Carrier frequency: 1 in 20 - 25 (N. Europe).

The CFTR Gene

• Cystic fibrosis transmembrane conductance regulator gene (CFTR).
• Located on chromosome 7q31.2.
• 27 exons, spanning approximately 190 kb of genomic DNA.
• Approximately 6 kb cDNA, encoding a protein of 1,480 amino acids.
• Over 2,000 pathogenic variants identified so far.

Carrier Frequency by Ethnicity

• African: 1 in 85
• Ashkenazy Jewish: 1 in 29
• Bahraini: 1 in 36
• Mexican: 1 in 46

Molecular Testing

Common CF Mutations

• Over 2,000 mutations identified.

Mutation Analysis

Molecular Testing: Multiplex Tests

• How many mutations to test?
  • F508delFAM: Only tests for p.F508del
  • CF4: Four most common mutations in UK (ΔF508, G542X, G551D, 621+1G>T)
  • CF-EU2: Panel of 50 most common mutations

F508delFAM Test

• Fluorescent PCR amplification of a region of exon 11 of CFTR.
• An allele with p.F508del will produce a fragment 3bp smaller than a normal allele.

CF4 Test

• Tests for 4 mutations, showing homo-/heterozygosity, based on ARMS (amplification refractory mutation system).
• For each mutation, primer specific to mutation (blue) and primer specific to normal allele (green) used.

CF-EU2 Test

• Tests for 50 mutations, showing homo-/heterozygosity.
• Based on ARMS (amplification refractory mutation system).
• For each mutation, primer specific to mutation (blue) and primer specific to normal allele (green) used.
• Primers split into two mixes:
  • “A mix” – primers to detect all mutations & p.F508 normal allele
  • “B mix” – primers to detect all normal alleles
• PCR followed by fragment analysis.

CF-EU2 Test Results

• Examples show results indicating no mutation, compound heterozygous (p.Gly551Asp(;) Arg1066Cys), and heterozygous for p.Phe508del.

p.Phe508del Mutation

• Most common CF mutation: c.1521_1523delCTT ® p.Phe508del [ΔF508]
• Approximately 70% of all CF mutations in NW European populations
• p.Phe508del = deletion of a phenylalanine (Phe) residue at position 508 in CFTR
• c.1521_1523delCTT = deletion of ‘CTT’ at positions 1521-1523 in CFTR

Mutation Analysis: Brown Family Example

• The analysis of common mutations detected only 1 heterozygous mutation p.Phe508del in Joanne’s sample
• Complete analysis of CFTR exons by sequencing identified two other variants: c.236G>A (p.Trp79*) and c.2620-15C>G which is close to the 3’ acceptor splice site of intron 15.
• We expect homozygous or compound heterozygous mutations in CFTR.

Routine Referrals to Molecular Testing

• To “rule out” CF in the newborn
• To confirm CF in suspected cases (sweat test positive)
• To determine carrier status of relatives of CF probands or known carriers
• To screen, e.g., for partners of carriers, egg/sperm donors
• To test after foetal echogenic bowel on ultrasound scan
• To analyze “monosymptomatic” forms of CF (= CFTR related disorders)
• To offer targeted therapy: some newly developed drugs require the precise mutation to be identified

Screening

Neonatal Screening

• Newborn screening for CFTR mutations in the UK is based on elevated immunoreactive trypsin (IRT) levels

Mutation Classes

Genotype/Phenotype Correlation

• Not all cystic fibrosis patients are the same
• There is significant clinical heterogeneity and the phenotype is variable.
• Different types of mutations:
  • in different domains of the gene/protein
  • associated with various gene modifiers
• This can give rise to a wide spectrum of MINIMAL to SEVERE disease

CFTR Protein Domains

• CFTR is a chloride channel in the apical membrane of secretory cells
• CFTR has 5 major domains:
  • two transmembrane domains (MSD)
  • two nucleotide-binding domains (NBD)
  • one regulatory domain (RD)
• Phosphorylation of RD controls ATPase activity of NBD and Cl− channel opening.

Drug Targets

• The identification of the different classes of mutations in CFTR has strongly contributed to the development of causative therapies using chemical drugs.
• Based on the pathologies of different mutations in CFTR, 4 major classes of mutations can be differentiated

Mutation Classes (I-IV)

• Functional differences have consequences for the development of therapies

Class I: Defective or Reduced Protein Production

• Include nonsense, frameshift, and splice site mutations, e.g., p.Gly542, p.Glu60, p.Glu56Aspfs
• Give rise to premature termination signals, leading to unstable transcripts or aberrant proteins
• No naturally occurring aberrant proteins have been described
• Loss of chloride channel activity

Class II: Defective Processing

• Majority of CF mutations, including p.Phe508del
• Give rise to a translation product unable to fold in the same way as normal.
• p.Phe508del CFTR does not escape the ER, and trafficking is therefore disrupted; protein does not reach the cell membrane.
• Under certain conditions, p.Phe508del CFTR can reach the cell membrane where it has some residual chloride channel activity.

Class III: Defective Gating

• Many mutations of NBDs, including severe mutations, such as p.Gly551Asp, and less severe mutations, e.g., p.Ala455Glu and p.Pro574His
• Interfere with binding of ATP or with ATP stimulation
• Result in a decrease in chloride channel activity
• p.G551D and p.G1349D affect ATP binding and inhibit channel opening.
• p.F508del, a class II mutation, also leads to changes in the NBD and a decrease in channel opening capacities if the mutant protein is guided to the membrane location.

Class IV: Defective Conduction

• Include mutations located in the pore, e.g., p.Arg117His, p.Arg334Trp, p.Arg347Pro
• Most mutations are in the MSD
• Normal phosphorylation and ATP-dependent regulation, but reduced single-channel currents – some mutations reduce the length of time the channel is open.

Functional Classes of CFTR Mutations

Biological and Genetic Therapies

Therapeutic Approaches

CFTR Modulator Therapies

• Potentiator
• Corrector and potentiator
• Read-through modulator
• Amplifier

Protein Repair – Ataluren (PTC124) [discontinued]

• Promotes read-through of premature stop codons such as p.Gly542* (class I mutation).
• Studies on p.Trp1282* in Israel and USA had promising results (p.Trp1282*: class I; 60% of CF mutations in Ashkenazi Jews).
• Phase 3 trial completed but trial discontinued › did not meet its primary and secondary endpoints of change in lung function and rate of pulmonary exacerbations
• Translarna® (ataluren) has been approved by the European Medical Agency (EMA) for DMD

Protein Repair – ELX-02 [ongoing]

• Promotes read-through of premature stop codons such as p.Gly542* (class I mutation).
• A phase 2 study to test the safety and tolerability of ELX-02 is currently underway
• The study is for people with CF who have at least one copy of the p.Gly542* mutation.

Protein Transport – Lumacaftor (VX809) [approved]

• Lumacaftor (VX809) is a corrector that acts as a chaperone to deliver defective CFTR to the cell membrane, for instance, CFTR:p.Phe508del (class II mutation).
• Orkambi® (lumacaftor + ivacaftor) has been approved 2015 by the US Food and Drug Administration (FDA) and the EMA for CF patients with two copies of p.Phe508del aged 2 years and older

Protein Assistance – Ivacaftor (VX770) [approved]

• Ivacaftor (VX770) acts as a potentiator, shown to increase gating activity.
• Studies carried out using p.Gly551Asp and p.Gly1349Asp (class III mutations).
• Up to 10% increase in lung forced expiratory volume (FEV1); improved nasal potential difference.
• Kalydeco® (ivacaftor) has been approved 2012 by EMA and FDA for CF patients with certain class III and IV mutations aged 4 months and older.

Combination Drugs

• Symkevi®/Symdeko®: Tezacaftor and ivacaftor
  • Corrector & potentiator. EMA and FDA approved 2018 for patients with two copies p.Phe508del or one copy of p.Phe508del and one copy of certain class III and IV mutations aged 6 years and older. (Class II, III, and IV)
• Orkambi®: Lumacaftor and ivacaftor
  • Corrector & potentiator. EMA and FDA approved 2015 for patients with homozygous p.Phe508del mutations aged 2 years and older
• Kaftrio®/Trikafta™: Elexacaftor, tezacaftor and ivacaftor
  • 2 correctors & 1 potentiator. EMA and FDA approved 2020/2019 for patients with at least one Phe508del mutation aged 12 years and older (Class II, III, and IV)

Protein Amplification – Nesolicaftor (PTI-428) [discontinued]

• Nesolicaftor (PTI-428) acts as an amplifier, shown to increase the amount of CFTR protein in the cell.
• PTI-428 can improve lung function in CF patients already receiving Orkambi®.
• Phase 2 studies were conducted to evaluate a combination therapy of PTI-428 with two modulators in patients with either one or two copies of p.Phe508del
• PTI-428 increased CFTR protein expression in nasal mucosa but did not have a significant impact on lung function. No further studies are planned at this time.

Other Therapies: Gene Therapy

• Gene Supplementation Therapy using a correct version of CFTR - transfected or transduced into the respective target cells. Note that the DNA has to enter the nucleus to be transcribed, which is a major barrier in gene therapy.
• Transcript Supplementation Therapy using a correct version of CFTR-mRNA transfected into the respective target cells. Note the mRNA is actively producing CFTR already in the cytoplasm, thereby circumventing the nuclear membrane.
• Protein supplementation therapy using a correct version of CFTR - transfected or transduced into the respective target cells. Note that protein delivery is often ineffective and it is difficult to include all natural post-protein-modifications.

Viral Vectors

• 4D-710 is a customized adeno-associated virus (AAV) vector therapy designed to deliver CFTR specifically to cells in the lungs (phase 1 trial)
• Possible unexpected side effects

Single molecules of nucleic acid compacted into nanoparticles (advantages?)

• No immune response or significant side effects
• transfer transient (6 - 28 days) and efficiency moderate but increased if delivered with liposomes
• VX-522 is an inhaled mRNA therapy. It aims to deliver a full-length copy of CFTR mRNA to lung cells using a lipid nanoparticle (phase 1 trial).

Gene editing

Need to reach the airway stem cells

Summary: Key learning points

• Cystic fibrosis is a pleiotropic disease with extensive allelic heterogeneity and some genotype/phenotype correlation.
• Distribution of mutations depends on populations:
  • Only a few different mutations account for more than 90% of CF chromosomes in N/W/C Europe.
• Mutations in CFTR can be assigned to distinct functional classes based on their pathomechanism.
• Causal pharmacological therapy is promising: Various drugs, including small molecules, are being developed for specific mutation classes.